The long term objective of the proposed research is to develop a clinically useful measure of tumor hypoxia. Tumor hypoxia can compromise the effectiveness of radiation treatment because of the well-known radioresistance of hypoxic cells. Tumor hypoxia is also seen as a target for selective chemotherapy with hypoxic cell cytotoxins. Rational intervention directed at hypoxic cells using hyperbaric oxygen, hypoxic cell radiosensitizers or hypoxic cell cytotoxins is confounded by the absence of a practical way of measuring hypoxia in individual tumors in particular patients. This grant proposes that hypoxic cell markers can serve this purpose. In the absence of oxygen, cellular redox enzymes activate nitroaromatic compounds in a way which leads to their irreversible binding to cellular proteins. The bound molecules becomes markers of cellular hypoxia. When suitably labelled, the markers can be detected by autoradiography, gamma ray scintigraphy, positron emission tomography, magnetic resonance spectroscopy or immunohistochemistry. In the immunohistochemical approach proposed, fluorescent antibody reagents clamp onto a marker molecule bound to cellular protein. The presence of hypoxia in sections of tumor tissue is then revealed upon fluorescence microscopic examination. The feasibility of using this approach to measure tumor hypoxia has been established in preliminary studies. The specific aims of the proposed research include optimizing the immunohistochemical approach for application in tumors of canine patients with a view to establishing a basis for early clinical application of immunohistochemistry to the measure of tumor hypoxia on an individual tumor basis.